Purine-2(3H),6(1H)-dione

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E 729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 99.9%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal concentrations selected for study were 1.3, 2.5, 5.0, 10 and 20 mg a.s./L; when measured concentrations of samples were averaged, geometric mean measured test concentrations for study were 0.28, 0.37, 1.7, 4.7 and 15 mg a.s./L, representing 21, 15, 35, 47 and 73% of nominal concentrations, respectively (results of study based on geometric mean measured concentrations).
- Sampling method: Duplicate test water samples were collected from batches of test solution prepared for each treatment and control group at beginning of test, and from two of four replicate test chambers in each group at 48 hours (± 1 hour) to measure concentrations of test substance.
- Sample storage conditions before analysis: room temperature

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test concentrations were adjusted for active ingredient of test substance based on reported purity of 99.9% (per CoA); individual test solutions prepared by weighing calculated amounts of test substance into containers; test solutions sonicated for 15 minutes and stirred on a magnetic stir plate for approximately 30 minutes. After mixing, test solution in 5.0 mg a.s./L treatment group appeared clear and colorless with no evidence of precipitation. The test solutions in 10 and 20 mg a.s./L treatment groups appeared clear and colorless with precipitate throughout water column and Tyndall effect was observed.
- Name of vehicle: water
- Controls: Negative control group was dilution water only with no test material.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 5.0, 10 and 20 mg a.s./L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Cladoceran
- Strain/clone: Daphnia magna
- Source: Eurofins
- Justification for species other than prescribed by test guideline: Representative of important group of aquatic invertebrates and was selected for use in test based upon history of use in laboratory.
- Age at study initiation (mean and range, SD): Parental stock- age not provided; < 24-hour old neonates obtained from cultures used in testing.
- Method of breeding: Four adult daphnids (used to supply neonates for test) held at least 10 days prior to collection of neonates; each produced at least one previous broods by Day 12 of holding; adult daphnids in culture had produced an average of at least three young per adult per day over 7-day period prior to test.
- Feeding during test: Daphnids in cultures fed daily; Adults fed during 24-hour period prior to test initiation, but neonates not fed during test.

ACCLIMATION
- Acclimation conditions: Same as in testing
- Type of food: Mixture of yeast, cereal grass media and trout chow (YCT), supplemented with vitamin stock solution and suspension of freshwater green alga, Raphidocelis subcapitata.
- Feeding frequency: Adults fed during 24-hour period prior to test initiation; frequency not provided.
- Health during acclimation (any mortality observed): Adults showed no signs of disease or stress, no ephippia were produced during holding period, and immobility in culture stock was 0% in two-day period prior to test initiation.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

148 mg/L as CaCO3

Test temperature:

Conducted at target water temperature of 20 ± 1°C.

pH:

8.0 - 8.3

Dissolved oxygen:

≥ 7.8 mg/L (≥86% of air saturation)

Salinity:

188 mg/L as CaCO3

Conductivity:

315 uS/cm

Nominal and measured concentrations:

0.28, 0.37, 1.7, 4.7 and 15 mg a.s./L

Details on test conditions:

TEST SYSTEM
- Test vessel: exposure chambers
- Type (delete if not applicable): open
- Volume of solution: 200 mL
- Aeration: yes
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: Loading rate was one daphnid per at least 20 mL of test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: freshwater obtained from well approximately 40 meters deep located on Eurofins site; well water passed through sand filter and pumped into a 37800-L storage tank where water was aerated with spray nozzles; prior to use in test system, water was filtered to 0.45 µm to remove fine particles and passed through an ultraviolet (UV) sterilizer.
- Total organic carbon: <1 (N=1)
- Metals, Organics, Pesticides: periodic analyses performed; In addition, specific conductance, hardness, alkalinity, pH and total organic carbon (TOC) content measured.
- Culture medium different from test medium: no
- Intervals of water quality measurement: Duplicate test water samples collected from batches of test solution prepared for each treatment and control group at beginning of test, and from two of four replicate test chambers in each group at 48 hours (± 1 hour) to measure concentrations of test substance; samples (5.0 mL) collected from mid-depth; one set of samples collected at each sampling interval processed immediately for analysis (duplicate set of samples stored under refrigerated conditions as back-up samples for possible later analysis).

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 6 hours of light and 8 hours of darkness.
- Light intensity: illuminated using fluorescent tubes with 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in light intensity.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Daily observations of immobility and clinical signs of toxicity.

VEHICLE CONTROL PERFORMED: yes (dilution water)

RANGE-FINDING STUDY
- Test concentrations: 0.16, 0.80, 4.0, 20 and 100 mg a.s./L (48 hours under static test conditions)
- Results used to determine the conditions for the definitive study: Percent immobility in negative control and 0.16, 0.80, 4.0, 20 and 100 mg a.s./L treatment groups at test termination was 0, 0, 0, 0, 0 and 10%, respectively.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks:

Lack of immobility at highest concentration tested, which was at limit of water solubility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 15 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks:

Lack of immobility at highest concentration tested, which was at limit of water solubility

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: None
- Other biological observations: Highest nominal concentration causing no immobility at test end was 20 mg a.s./L and the lowest nominal concentration causing 100% immobility at test end was > 20 mg a.s./L.
- Immobilisation of control: immobility in the control group was 0%. There also was no sign of disease or stress in the control group.
- Abnormal responses: None

MEASURED CONCENTRATIONS OF TEST SUBSTANCE IN FRESHWATER SAMPLES
Nominal Test Concentration (mg a.s./L)	Sample Number	Sampling Time (Day)	Measured Concentration^1,2 (mg a.s./L)	Percent of Nominal^2	Geometric Mean ± SD Measured Concentration ^3 (mg a.s./L)	Geometric Mean Measured Percent of Nominal^3
Negative Control (0.0)	1	0	<30% LOQ	--	<30% LOQ	--
	7	2	<30% LOQ	--
	8	2	<30% LOQ	--
1.3	2	0	1.39	1.07	0.28 ± 0.944	21
	9-RE^4	2	0.0246	1.89
	10-RE^4	2	0.0850	6.54
		Average:	0.0548	4.22
2.5	3	0	2.44	97.5	0.37 ± 1.69	15
	11-RE^4	2	0.0737	2.95
	12-RE^4	2	0.0359	1.44
		Average:	0.0548	2.20
5.0	4	0	4.69	93.7	1.7 ± 2.86	35
	13	2	0.820	16.4
	14	2	0.460	9.20
		Average:	0.640	12.8
10	5	0	8.66	86.6	4.7 ± 4.34	47
	15	2	2.50	25.0
	16	2	2.53	25.3
		Average:	2.52	25.2
20	6	0	18.1	90.3	15 ± 4.38	73
	17	2	11.0	54.8
	18	2	12.7	63.5
		Average:	11.9	59.2
^1The limit of quantitation (LOQ) of test substance in freshwater is set at 0.100 mg a.s./L, defined as the lowest nominal concentration of matrix fortification samples with a mean recovery within 70-120% and a relative standard deviation (RSD) ≤ 20%. The limit of detection (LOD) of test substance in freshwater is set at 0.0200 mg a.s./L, defined as the lowest detectable concentration of an analyte in a sample and expressed as the lowest calibration standard concentration (0.00500 mg a.s./L) multiplied by the dilution factor of an LOQ sample (4.00). ^2Results were generated using Analyst Version 1.7.1. Manual calculations may differ slightly. ^3Results were generated using Excel 2016. Manual calculations may differ slightly. ^4Original results had peak areas below the calibration curve thus reanalysis was done. Reanalysis results were reported.

 

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 value for the test substance was > 15 mg a.s./L, the highest concentration tested, which was at limit of water solubility.

Executive summary:

The acute toxicity of the test material to Daphnia magna under static conditions was determined in a 48-hour exposure test. The test was conducted based on procedures in the OECD Guidelines for Testing of Chemicals, Guideline 202: Daphnia sp., Acute Immobilization Test; the U.S. EPA Series 850 – Ecological Effects Test Guidelines, OCSPP 850.1010: Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids; and ASTM Standard E 729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians. Daphnids were exposed to a geometric series of five test concentrations and a negative control (dilution water) for 48 hours under static conditions. Four replicate test chambers were maintained in each treatment and control group, with five daphnids in each test chamber, for a total of 20 daphnids per concentration. Neonate daphnids < 24-hours old were impartially assigned to exposure chambers at test initiation. Observations of immobility and other signs of toxicity were made approximately 3.5, 24 and 48 hours (± 1 hour) after test initiation. Cumulative percent immobility observed in the treatment groups was used to determine EC50 values at 24 and 48-hour intervals. The 48-hour NOEC, the highest nominal concentration causing no immobility at test end, and the lowest nominal concentration causing 100% immobility at test end were determined by visual interpretation of the immobility data. Nominal test concentrations selected were 1.3, 2.5, 5.0, 10 and 20 mg a.s./L. Test concentrations of the test substance were measured in samples of test water collected from each treatment and control groups at the beginning and end of the test using high performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS). Results of the analyses were used to calculate geometric mean, measured test concentrations.
Measured concentrations of the samples ranged from approximately 86.6 to 107% of nominal at the beginning of the test and from approximately 1.44 to 63.5% of nominal at the end of the study. When measured concentrations of the samples were averaged, the geometric mean measured test concentrations for this study 0.28, 0.37, 1.7, 4.7 and 15 mg a.s./L, representing 21, 15, 35, 47 and 73% of nominal concentrations, respectively. The results of the study were based on the geometric mean measured concentrations.
At the end of the exposure, all daphnids in the control group and all the treatment groups appeared normal throughout the test with no signs of stress. Therefore, in this study, highest nominal concentration causing no immobility at test end was 15 mg a.s./L and the lowest nominal concentration causing 100% immobility at test end was >15 mg a.s./L. Lack of immobility in any of the treatment groups precluded statistical evaluation of EC50 value and NOEC value. Therefore, the 48-hour EC50 value was empirically estimated to be >15 mg a.s./L (highest concentration tested which was at limit of water solubility). The 48-hour NOEC was determined to be 15 mg a.s./L.

Description of key information

OECD Guideline 202; Daphnia magna 48-hr EC50 >15 mg a.s./L (highest concentration tested which was at limit of water solubility). Reliability = 1

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 15 mg/L